Various methods are in use for the detection and measurement of biological materials. To date, these determinations are generally facilitated through the use of radiological, fluorescent or enzymatic tags. None of these methods have successfully dealt with elemental tagging of biologically active materials, for example immunoglobulins, aptamers or antigens, and analytes followed by detection using atomic mass or optical spectrometry.
The methods used to date have included (1) elemental tagging in immunoassays, (2) elemental tagging using radioisotopes, (3) elemental tagging to enhance fluorescence, (4) immunological detection of elemental species without tagging, and (5) direct elemental tagging for cell uptake studies. We will review each of these areas in turn.
Elemental Tagging in Immunoassays
Wang 1984 (U.S. Pat. No. 4,454,233) disclosed the possibility of utilizing a mass spectrometer as a means of immunoassay detection. Wang's method required a cumbersome preliminary set of steps to first prepare a tagged ‘mobile unit’ which was then conjugated to an antibody/antigen. In the preferred embodiment, the ‘mobile unit’ was comprised of a latex particle embedded with heavy tagging elements such as Fe, Ni, Cu and Co. Among Wang's reasons for utilizing ‘mobile units’ were: (1) easy separation of bound reactant from unbound reactant, (2) simultaneous detection of many antigen/antibody complexes owing to the small size of mobile units, and (3) possible utilization of ‘unstable’ or ‘reactive’ tags, as tags embedded in the latex would not interfere with the reaction. Wang's method has apparently not been accepted for immunoassay detection.
Immunoassay detection using element tagged immunoglobulins and antigens has also been possible using colloidal gold or extremely small beads of gold (several nanometers in diameter), for example NANOGOLD (registered trademark of Nanoprobes Inc) particles. Van Banchet and Heppelmann (1995), Wagenknecht et al. (1994), and Wenzel and Baumeister (1995) used colloidal gold to visualize protein structure in the cell and to detect receptor-ligand binding by electron microscopy. However, these assays suffer from lack of quantitation capabilities.
Element tagging has also been used in electrochemical immunoassays followed by polarographic detection of the generated complexes based on the catalytic conversion of a substrate by labeled metal ion or the anodic current of metal labeling Qiu and Song (1996). Similar to the preceding example, the assay lacked quantitation capabilities.
Thus, although elemental tagging has been used in immunoassays, the tagging methods have been cumbersome or were ineffective at quantitation.
Elemental Tagging Using Radioisotopes
Historically, the most common use of elemental tagging has been the use of radioactive elements. While radioassays remain the method of choice due to their exceptional sensitivity to low levels of analyte, their general use is limited by the restrictions in dealing with radioactive materials.
Elemental Tagging to Enhance Fluorescence
Recently, elemental tagging has been used to enhance luminescence of fluorescent tags. U.S. Pat. No. 4,637,988 to Hinshaw et al., describes the use of lanthanide metals complexed with fluorescent compounds and chelating agents, that can be used in specific binding assays. U.S. Pat. No. 5,958,783 to Josel et al. describes the use of metal complexes with a charged linker as luminescent groups in fluorescence-based or electrochemiluminescence-based assays.
However, these fluorescent tagging methods suffer from the disadvantages associated with their relatively low sensitivity and resulting problems with quantitative analysis in samples containing low concentrations of target molecules. As well, fluorescence-based methods are limited to the analysis and quantification of only one or at most a few target substances per assay.
Immunological Detection of Elemental Species
Blake et al. (1998) and Darwish and Blake (2001) disclosed a method of detection and quantitation of elemental species by complexing elemental species with antibodies that recognize elemental species, using antibodies conjugated with fluorescent tags. However, as outlined above, fluorescence based assays suffer from low sensitivity and are limited to one or a few targets per assay.
Direct Elemental Tagging in Conjunction with Gel Electrophoresis
Binet et al. (2001) disclosed a method of determining untagged proteins by separation using gel electrophoresis, followed by laser ablation of the separated spots and detection using mass spectrometry. However, this method has been limited to molecules that are naturally detectable by spectrometry.
Wind et al. (2001), Nagaoka and Maitani (2000) and Baldwin et al. (2001) used chromatography to separate proteins, followed by detection using mass spectrometry. Similarly, Chen et al, 2000 incorporated isotopic tags of 13C, 15N and 2H in proteins before chromatographic separation and detection using organic mass spectrometry. However, separation with chromatography is an added step, which can be onerous.
Thus, if one does not tag, one is limited to what is being assayed and using chromatography for separation adds a step to the process.
Direct Elemental Tagging for Cell Uptake Studies
Martin de Llano et al. (1996) and Martin de Llano et al. (2000), disclosed a method of visualizing and measuring the uptake of low density lipoprotein (LDL) tagged with colloidal gold by cells using electron microscopy and mass/atomic spectrometry. However, due to the use of colloidal particles, the difficulty in purifying labeled LDL, and the heterogeneity of cell assay systems, absolute quantitation was not necessary nor was it achieved.
Thus, various methods have been developed for visualizing and analyzing element tagged biologically active compounds. However, they have innate limitations, ranging from handling radioactive waste, to low sensitivity with fluorescence based assays, to detection only capabilities, and to cumbersome preparation or separation steps.
Various kits are currently in use for the detection and measurement of analytes. These kits contain radiological, fluorescent, or enzymatic reagents and are used in conjunction with detection instruments capable of measuring absorbance, luminescence, fluorescence, chemiluminescence, or radioactivity. However, none of these kits or methods have successfully dealt with the detection and measurement of element tagged biologically active materials and analytes, in particular immunoglobulins, aptamers, and antigens, followed by detection and measurement using an atomic mass or optical spectrometer.
Reagents (not currently sold in a kit format) containing element tagged immunoglobulins are commercially available (NanoProbes). In these reagents, the immunoglobulins are directly bound with colloidal gold or extremely small beads of gold (eg. NANOGOLD particles, which are 1.4 nm in diameter) and are currently used for in situ hybridization, electron microscopy and immunohistochemistry. In this manner, Segond von Banchet and Heppelmann (1995) and Wagneknecht et al. (1994) used colloidal gold to visualize protein structure in the cell and to detect receptor-ligand binding by electron microscopy. In another element-tagged method, Leuvering et al. (1982) have suggested using large elemental particles (with a size varying from 10-100 nm) coated either directly on immunological components or on inert polymer linkers and using spectrophotometric detection to analyze reactions. However, both of these assays suffer from lack of quantitation capabilities.
Recently, in immunoassay kits, elemental tags have been used to enhance the luminescence of fluorescent tags of immunoglobulins. Hinshaw et al. (1987) describe the use of lanthanide metals complexed with fluorescent compounds and chelating agents that can be used in specific immunoassays. Josel et al. (1990) describe the use of metal complexes with a charged linker as luminescent groups in fluorescence-base or electrochemiluminescence-based assays. However, these fluorescent lanthanide tag kits suffer from the disadvantages associated with their relatively low sensitivity and resulting problems with quantitative analysis in samples containing low concentrations of target molecules. As well, fluorescence-based methods are limited to the analysis and quantitation of only one or at most a few target substances per assay.
Several companies have designed kits for cytokine quantitation that contain radiological, fluorescent, or enzymatic reagents. For example, PerkinElmer (Wallac DELFIA (registered trademark of Wallace Oy) Assay kits), BD Biosciences (OptEIA ELISA (registered trademark of Becton, Dickinson and Company) kits), Pierce Biotechnology Inc. (Searchlight Human Cytokine (registered trademark of Pierce Biotechnology Inc) array), R&D systems (Quantikine ELISA (registered trademark of Research & Diagnostic System, Inc) kits) and various partners of Luiminex (e.g. R&D systems, Fluorokine kits) produce kits for cytokine quantitation. However, these kits are limited to either detecting only one cytokine or several (4-9) over a limited dynamic range with problems of fluorescence or chemiluminescence overlapping inhibiting sensitivities.
An enormous potential exists for the development of very simple biological assays and kits that take advantage of capabilities offered by elemental tagging coupled with elemental detection using a mass or optical spectrometer. Mass and optical spectrometry offer high sensitivity, accurate quantitation and a wide dynamic range. The use of elements to label biologically active material allows construction of an enormous number of distinguishable tags.
This invention involves bridging the science of biology, and in particular immunology, with analytical atomic mass spectrometry. The invention offers an easy and simple means of tagging biological molecules. Further, it offers excellent detection capabilities, equaling (or surpassing) the sensitivity of radioassays. It offers the safety of florescence based assays, and the added feature of an enormous number of available tags, with the possibility of simultaneous detection of numerous biological complexes. In addition, completed affinity assays can be stored indefinitely and the handling of the reacted tagged complexes can be crude, as the integrity of the chemical complex need not be preserved in assaying the element.